PROJECT SUMMARY Endogenous homeostatic mechanisms can restore normal neuronal function following cocaine-induced neurotoxicity. Such mechanisms may be exploited in developing novel therapies for cocaine addiction, but a molecular target has not yet been identified. Here we profiled mouse gene expression in the nucleus accumbes (NAc), during early and late cocaine abstinence to identify putative regulators of neural homeostasis. Cocaine regulated expression of the transcription factor, Nuclear Receptor Subfamily 4 Group A Member 1 (Nr4a1), and its target gene, cocaine and amphetamine-regulated transcript peptide (Cartpt), a key molecule in mitigating dopamine toxicity. Sustained activation of Cartpt at late abstinence was coupled with depletion of the repressive histone modification, H3 lysine 27 tri-methylation (H3K27me3), and enrichment of activating mark, H3 lysine 27 acetylation (H3K27ac). Using both CRISPR-mediated and small molecule Nr4a1 activation, we demonstrate the direct causal role of Nr4a1 in this epigenetic mechanism of sustained gene expression and in repression of cocaine-evoked behavior. Our findings provide evidence that targeting endogenous changes in homeostatic gene expression across abstinence is a potential strategy to combat cocaine addiction. The overall goal of this study is to determine the pathways responsible for observed changes in Nr4a1 and target gene expression by isolation of tagged nuclei in specific cell types (INTACT). To this end, we will measure cocaine-induced gene expression and histone post translational modifications in direct (D1-expressing) and indirect pathway (A2A-expressing) medium spiny neurons (MSNs) during abstinence. Additionally, will extend our findings using contingent models of drugs of abuse, namely, cocaine self-administration to evaluate Nr4a1 epigenetic regulation of target genes in male and female mice. Finally, we will determine co-factors involved in the epigenetic regulation of Nr4a1 target genes. Taken together, this proposal will improve our understanding of the precise epigenetic mechanisms by which individual genes ?remember? prior drug exposure.